Design and development of turn-on fluorescent probes for detecting carbapenemases in β-lactam resistant bacteria

Abstract

In this study, an activity-based fluorogenic probe for carbapenemase detection was first designed and synthesized. The probe provided more than 60-fold fluorescence enhancement together with chromogenic changes upon hydrolysis catalyzed by carbapenemases. It displayed good selectivity for carbapenemases over non-carbapenemases, and produced significant fluorescence enhancement upon incubation with OXA-48 carbapenemase which existing phenotypic assays failed to identify. The assay demonstrated high sensitivity, with the detection limit for carbapenemases in picomolar to sub-picomolar range. Michaelis–Menten kinetics model was used to evaluate the carbapenemase-substrate efficiency. The carbapenemases were found to have kcat/KM > 106 M-1s-1, suggesting the probe serves as a good general substrate towards a range of carbapenemases. The probe performance was then evaluated with clinical isolates. A rapid test protocol was then optimized and a direct comparison was made under similar conditions with existing CarbaNP chromogenic test in the presence of clinical isolates. The probe outperformed CarbaNP, with obvious color changes and fluorescent signals observed within 15 minutes. Using our probe, a carbapenem-resistant clinical isolate that has no carbapenemase expression was successfully detected. The defection of an AmpC encoded strain that is capable of hydrolyzing imipenem, demonostrates the power of our activity-based phenotypic test. Moreover, a protocol to detect carbapenem-resistant bacteria was successfully optimized by using LB broth culture, which reduced the time for bacterial culture by one day. In the later stage of the project, a number of derivatives was synthesized to improve the aqueous stability of our lead compound as well as to explore the enzyme-substrate scope. In particular, the carboxylic acid group was confirmed to be an indispensable feature for carbapenemase recognition. Meanwhile, installation of β-methyl substituent on carbapenem led to poor OXA-48 enzyme kinetics. High concentration AmpC displayed some hydrolytic activity towards one probe with β-methyl group replaced by a proton. These probes allow rapid tests in clinical setting for carbapenemase detection without any sophisticated instrument and expensive consumables. On the other hand, it may have many applications including identification of new carbapenemases and drug discovery (e.g. discovery of new carbapenemase inhibitors). The current study also provides a comprehensive structure-activity relationship study on carbapenemases, which would be useful for designing new carbapenem antibiotics.